Interspecific Mating Behavior Between Introduced Zacco platypus and Native Opsariichthys evolans in Taiwan

© 2020 Academia Sinica, Taiwan Open Access Interspecific Mating Behavior Between Introduced Zacco platypus and Native Opsariichthys evolans in Taiwan Neng-Li Liao1,2, Shih-Pin Huang1, and Tzi-Yuan Wang1,* 1Biodiversity Research Center, Academia Sinica, Nankang, Taipei 115, Taiwan. *Correspondence: E-mail: [email protected] (Wang). E-mail: [email protected] (Huang) 2Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung 807, Taiwan. E-mail: [email protected] (Liao) Received 9 October 2018 / Accepted 22 January 2020 / Published 9 March 2020 Communicated by Hin-Kiu Mok Introduced freshwater fishes considerably influence the ecology and populations of native species. Previous research has revealed that introduced Zacco platypus may hybridize with chubs that are sister but distinct genera. However, we have little knowledge of Z. platypus’ mate choice or its impact on Taiwanese chubs. Therefore, this study identified the interspecific mating behavior between introduced Z. platypus and native Opsariichthys evolans and evaluated the former’s invasive impact on cohabitants. Our observations showed that interspecific mating pairs do occur between Z. platypus male(s) and O. evolans female(s). Fifty-three percent of spawning events were interspecific mating and only 43% were between native O. evolans mating pairs. This study showed that Z. platypus male satellites might prefer to engage with Z. platypus, while O. evolans might engage by chance. However, introduced males of Z. platypus may be unable to recognize conspecific females. Meanwhile, introduced females of Z. platypus also have a mate choice preference for males of Z. platypus. Therefore, Z. platypus male hybridization might significantly reduce the successful mating ratio of O. evolans, leading to a dramatic reduction in native O. evolans offspring in the future. Key words: Spawning behavior, Chub, Mate choice, Opsariichthys, Zacco. Citation: Liao NL, Huang SP, Wang TY. 2020. Interspecific mating behavior between introduced Zacco platypus and native Opsariichthys evolans in Taiwan. Zool Stud 59:6. doi:10.6620/ZS.2020.59-06. BACKGROUND The Opsariichthys group (Teleostei: Cypriniformes) contains common minnows found in freshwater habitats, including the genera Parazacco, Candidia, Nipponocypris, Zacco, and Opsariichthys (Huang et al. 2017). There are five species in the Opsariichthys group in Taiwan—three endemic Opsariichthys species, Candidia barbata (Regan, 1908), and the introduced Zacco platypus (Temminck & Schlegel, 1846) (Chen et al. 2008 2009; Huang et al. 2017; Kitanishi et al. 2016; Ma et al. 2006). Zacco platypus was identified in the Tamsui River as an introduced species from Japan in the 1980s (Ma et al. 2006). Its D-loop nucleotide divergence among haplotypes ranged from 0.2% to 2.82%, and its high haplotype diversity implies multiple introduced populations from Japan. The origin of Z. platypus in Taiwan is questionable, although a previous study suggests that it came from Lake Biwa, Japan (Ma et al. 2006). Nevertheless, the introduced Z. platypus may greatly influence closely-related native Opsariichthys species. Introduced freshwater fishes have considerably influenced the ecology and populations of native species. However, we have little knowledge on Z. platypus’ mate choice or impact on Taiwanese chubs, although a similar mating behavior and reproductive process in chubs was reported in Japan (Katano 1985 1990a b 1992 Zoological Studies 59: 6 (2020) doi:10.6620/ZS.2020.59-06 1 © 2020 Academia Sinica, Taiwan 1994 1998) and Taiwan (Chuang et al. 2006; Wang et al. 1995; Yan et al. 1995). For example, the mating behavior of Candidia barbata was reported as being similar to that of the Japanese dark chub Nipponocypris temminckii (Yan et al. 1995). Their spawning behaviors are quite similar to those of N. temminckii: a male presses a female laterally and stirs up the riverbed by vibrating its anal fin to bury released eggs (Katano 1983). Katano (1983) indicated two clearlydistinguished levels of social status in males: leading males that monopolize females and satellite males (sneakers) that dash toward spawning pairs. Leading males were the dominant individuals, but not always the largest. In some cases, they established territories around the spawning redd and, on one occasion, two leading males used the same spawning redd without establishing territories. In addition, two sympatric Japanese chubs—Z. platypus and Nipponocypris temminckii—are known to have aggressive interactions during their reproductive activities (Katano 1994). These two species commonly occupy overlapping home ranges and occasionally defend a territory against other individuals. Furthermore, the hybrids of Z. platypus and phylogenetically distinct Nipponocypris species—N. sieboldii (Temminck & Schlegel, 1846) and N. temminckii (Temminck & Schlegel, 1846)—have been reported, which might be due to artificial propagation, environmental temperature, and/or sympatric habitats increased by hydraulic engineering (Arao and Shimoyama 2006; Katano et al. 2014; Kato 2004; Sakai et al. 1992). Thus, it is very possible that the introduced Z. platypus hybridizes with Taiwanese Opsariichthys species, especially the cohabitated Opsariichthys evolans (Jordan & Evermann, 1902). This study identified such interspecific mating behaviors and evaluated the former’s invasive impact on cohabitants. There are several distinguishing morphological features between Z. platypus and O. evolans—different length of pectoral fin ray, different patterns of pearl organs, and different body color patterns between the males; different stripe colors and snout tip colors between the females (details see Fig. 1; MATERIALS AND METHODS). A recent study further revealed that Z. platypus females choose males based on the degree of their red nuptial coloration, which suggests that different body color patterns may influence mate choice (Takahashi 2018). Interspecific mating behavior has only been systematically studied in several species of animals, such as the leaf beetle, pipefish, and gecko (Peacock et al. 2004; Wilson 2006; Groning and Hochkirch 2008; Buden et al. 2014), and these cases were assigned as parts of reproductive activities due to misidentification, accident, mating, and/or stimulation. This study reveals interspecific mating behavior between introduced Z. platypus and native O. evolans and their mate choice; it shows how this interspecific mating may influence the population of native offspring by reducing the rate of O. evolans intraspecific mating. MATERIALS AND METHODS Study areas Two spawning areas with 7–19 redds in the Keelung River were chosen as the study areas; both are located in Ruifang District, New Taipei City in northern Taiwan. Study site A (25°06'22.8"N, 121°48'33.0"E) is about 4 m long and 3 m wide, and has an average depth of 15 cm. Study site B (25°06'22.9"N, 121°48'32.2"E) is about 8 m long and 5 m wide, and has an average depth of 13 cm. The water temperature is 26–36°C at both sites. Ethogram A successful spawning event is defined as follows: when spawning, both sexes remain close to each other and continually shake their caudal fins, then lean to one side and the female lays its eggs (Wang et al. 1995; Yan et al. 1995). This study defined the starting point of a spawning event as when both sexes shake their caudal fins (Table 1). After observing this shaking behavior, we continued to record the event until no other fishes appeared in the redd. Such a successful spawning event was counted as an independent event. Satellites Satellites are unpaired mature males (Table 1). These satellites always try to engage in cuckoldry to a mating pair during spawning. The paired male always followed with the paired female while the satellites swim around different mating pairs. Species identification There are several distinguishing morphological features between Zacco platypus and Opsariichthys evolans (Jordan & Evermann, 1902): different length of the pectoral fin ray, different patterns of pearl organs, and different body color patterns between the males (Fig. 1a, c); and different stripe colors and snout tip colors between the females (Fig. 1b, d). The body of O. evolans has a silver background with yellowish brown in the dorsal area, the body color page 2 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan patterns have 11–13 vertical greenish-blue stripes in mature males, and shorter stripes in females (Chen et al. 2009). The pectoral fin is rather long, and its maximum length reaches the pelvic fin base in mature males, but the pectoral fin is clearly shorter in females. Sexual dimorphism can be clearly observed during the reproductive season (Fig. 1a, b). Mature males have (1) independent pearl organs on the cheek and snout and (2) a yellowish green caudal peduncle; mature females do not have either character. In addition, parts of the pectoral, dorsal, and anal fins turn red in mature males, and these fins are usually yellow in mature females. The body of Z. platypus also has a silver background and is yellowish-brown in the dorsal area. But the body color patterns have several vertical grayish-blue stripes, some of which stripes fuse to form Table 1. Ethogram Behavior Definition Spawning event Both sexes remain close to each other and continually shake their caudal fins, then lean to one side and lay their eggs with disturbed sand. Cuckoldry Alternate male strategy; small males or satellite may engage in cuckoldry in species where spawning is dominated by large and aggressive males. Mating pair A large and/or aggressive male follows and cruises around a mature female during spawning. Territoriality During spawning, both sexes remain close to each other. The paired male circles around the redd and shows its fins to other approaching adults, and/or moves out to fight with the approaching individuals. Fig. 1. Character identification of two species. a, O. evolans male; b, O. evolans female; c, Z. platypus male; and d, Z. platypus female. All morphological features for species identification were labeled using Arabic numerals and mentioned as follows. The mature male of O. evolans has: 1, 11–13 vertical greenish-blue stripes; 2, elongated pectoral fin; 3, independent pearl organs on the cheek and snout; 4, yellowish green caudal peduncle. The mature female of O. evolans has: 5, shorter vertical stripes; 6, usually grayish white snout tip. The mature male of Z. platypus has: 7, several vertical grayish-blue stripes and some of these stripes fuse into a wide bar; 8, medium length of pectoral fin; 9, aligned pearl organs on cheek and snout. The mature female of Z. platypus has: 10, a rather indistinct pale yellow or gray stripe; 11, usually orange red snout tip. page 3 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan a wide bar in mature males. In addition, the stripes are a rather indistinct pale yellow or gray in females. Its pectoral fin is clearly shorter, and its maximum length does not reach the pelvic fin base. Sexual dimorphism can also be clearly observed during the reproductive season (Fig. 1c, d). Mature males have (1) aligned pearl organs on their cheek and snout, and (2) the tip of their snout is grayish black; mature females do not have pearl organs, and the tip of their snout is orange red. In addition, parts of the pectoral, ventral, dorsal, and anal fins turn red in mature males, and these fins are usually yellow in mature females. Females of the two species have fewer distinguishable characteristics than males (Fig. 1b, d). Nevertheless, two morphological features can still be distinguished between the two female chubs. First, females of both species have several vertical stripes, but those of O. evolans are yellowish green and those of Z. platypus are a rather indistinct pale yellow or gray. Second, the snout tip is usually grayish white in O. evolans females, but orange red in Z. platypus females. In addition, the pearl organs on the head and anal fin regions are also regarded as diagnostic features for distinguishing a mature male from a female and subadult individual. Katano (1990b) showed that pearl organs on the head are a weapon in conspecific aggressive encounters, and the pearl organ on the anal fin is a tool for burying eggs during spawning seasons; it is also highly associated with a large body size in males. Types of mating pairs Mature male and female chubs formed mating pairs during the spawning season (Table 1). The paired chubs then searched for a redd to spawn in. There were four possible types of mating pairs: (a) O. evolans♂ × O. evolans♀; (b) Z. platypus♂ × Z. platypus♀; (c) Z. platypus♂ × O. evolans♀; (d) O. evolans♂ × Z. platypus♀. The former two types were intraspecific and the latter two were interspecific. Furthermore, if satellites from different species engaged in the former two types, such a spawning event was also defined as interspecific mating. Video recording in habitats Digital handy cameras (Sony FDR-AXP55 and Cannon G16) and housing Polaroid CUBE+ cameras were used to record all mating behaviors. The spawning events were recorded in the studied habitat for five independent days from 9:00 to 12:00 in July 2018 (spawning season). The housing Polaroid CUBE+ cameras were placed near the spawning redds without disturbing the mating pairs (Fig. 2a, b, c). In addition, the digital handy cameras were also used to record chubs during the spawning event with a wider angle (Fig. 2d, e). We followed the paired male/female and recorded their spawning events via the digital handy cameras. All digital files were analyzed on a laboratory computer. To prevent the underwater housing cameras from interfering with the mating behavior, we first tested the working distance to the redd and determined whether the small camera was too close to the redd. Although mature males that spotted the camera sometimes tried to attack it with their snout or looked for a different nearby redd, the cameras did not affect the paired chubs’ spawning behavior in the redd (Fig. 2, video S1 and video S2). Statistics The types of mating pairs and number of satellites were counted independently within a successful spawning event. In addition, if the species of an individual could not be recognized, then the event was discarded in our statistics. The mate choice preference was determined by the Chi-square test of observed and expected spawning events. The Wilcoxon-MannWhitney rank sum test was used to determine if satellites had a significant preference for any of the three kinds of mating pairs. The Bonferroni multiple test was performed to evaluate if the false discovery rate (FDR) < 0.05. RESULTS Relative proportions of the two species To determine the putative proportions of offspring from different types of mating pairs in the two species, we calculated the relative proportions of the two species from observed mating pairs. The two supplemental videos show intraspecific and interspecific mating events recorded by the handy and housing cameras. We observed 78 spawning events over the spawning season, six of which were discarded because the species/ individuals could not be recognized. The 72 sampling events represented the types of mating pairs in our study. Of the individuals involved in the 72 recorded spawning events, 63 were male Z. platypus and 113 were male O. evolans (Table 2 and Table S1). On each recording day, the paired male followed the paired female. Thus, the mating pair number was represented by the relative proportion of introduced Z. platypus page 4 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan Fig. 2. The video clips from the housing Polaroid CUBE+ and handy cameras. (a) Intraspecific mating pair; (b) two paired chubs spawning nearby; (c) satellites trying to engage with paired chubs; (d) O. evolans mating; (e) Z. platypus mating. Clips (a–c) were captured on the housing camera. Clips (d– e) were captured on handy cameras. (a) (c) (d) (e) (b) Table 2. Total numbers of mating pair events and satellites Type of mating pair Spawning events Satellite Total number, involved events (%) Observed Expected O. evolans♂Z. platypus♂ O. evolans♂ × O. evolans♀39 (54%) 29.7 (41.2%) 37, 18 (45%) 9, 8 (38%) Z. platypus♂ × Z. platypus♀8 (11%) 9.2 (12.8%) 8, 5 (12%) 2,1 ( 5%) Z. platypus♂ × O. evolans♀24 (33%) 16.6 (23%) 28, 17 (43%) 20, 12 (57%) O. evolans♂ × Z. platypus♀1 (2%) 16.6 (23%) 0, 0 (0%) 0, 0 ( 0%) Total number 72 (100%) 72.1 (100%) 73, 40(100%) 31, 21(100%) page 5 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan (Zp) and native O. evolans (Oe) populations, which was around 35.8% and 64.2%, respectively. Based on this ratio, the expected frequency of Oe × Oe pairing under the assumption of random mating should be 0.642 × 0.642 = 41.2% (0.702 × 0.702 = 49.3%); Zp × Zp should be 0.358 × 0.358 = 12.8% (0.298 × 0.298 = 8.9%); and Oe × Zp and Zp × Oe should each be 23.0% (20.9%). Considering that the satellites still affect offspring number, we also used the relative proportion of satellites in each species (29.8% of Z. platypus vs 70.2% of O. evolans), which showed no significant difference in subsequent analyses (data not shown). There might, however, be some bias around the relative proportion of the species, such as putative multiple counts, uncounted female numbers, and environmental changes from human activities. In addition, we also harvested some specimens for future studies. The relative proportions of the species (Z. platypus vs O. evolans) ranged from 50.0% vs 50.0% to 11.1% vs 88.9% (Table S2). Such variation might be due to insufficient sampling size and/or seasonality. Nevertheless, the relative proportion of the two species (11.1% of Z. platypus vs 88.9% of O. evolans) during the spawning season was also used in subsequent analyses. Mating pairs Four types of mating pairs were observed, but their ratios were not even (Table 2). Our observations revealed that 35% of spawning events were from interspecific mating pairs. Only 54% of spawning events were from native O. evolans mating pairs. In addition, 8 of the 39 O. evolans spawning events were engaged by Z. platypus satellites; 5 of the 8 Z. platypus spawning events were engaged by O. evolans satellites. Thus, 53% of the spawning events were interspecific and only 43% were between native O. evolans mating pairs/satellites. Mating preferences Statistical analysis further revealed different mating preferences between males and females of both species, even when different relative proportions were used (Table 3). Males of O. evolans significantly preferred mating with females of O. evolans, while females only showed a slightly significant preference for males. In contrast, females of Z. platypus significantly preferred males of Z. platypus, across different relative proportions (Table 4). However, males of Z. platypus showed no significant preference for females of one species (Table 5), even though males of Z. platypus were shown to slightly prefer females of O. evolans when we used the capture ratio (11.1% of Z. platypus vs 88.9% of O. evolans) as a relative proportion (p-value = 0.0123, data not shown). Such a slight difference could be due to the uneven population proportion between the females of both species. Thus, males of Z. platypus might show no preference for females of either species. Table 3. Mate choice preference via Chi-square test of observed and expected spawning events O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀ Z. platypus♂ × O. evolans♀ O. evolans♂ × Z. platypus♀ O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀0.0784 Z. platypus♂ × O. evolans♀0.0122* 0.0613 O. evolans♂ × Z. platypus♀0.0000*** 0.0001*** 0.0000*** *p < 0.05, ***p < 0.005. Table 4. Mate choice of the two female chubs O. evolans ♀Z. platypus ♀ Observed expected observed expected O. evolans ♂39 40.45 1 5.78 Z. platypus ♂24 22.55 8 3.22 p-value 0.7039 8.93E-04**** ****p < 0.001. page 6 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan Satellite male engagement preference between the species Table 6 indicates the number of satellite males that joined the mating process and table 7 presents the number of mating cases that satellite males engaged in. Tables 8 and 9 further show that when the mating female is O. evolans, Z. platypus and O. evolans satellites prefer to engage in mating pairs with a male of the same species, even using different relative proportions. The satellites did not show any difference in preference in the other comparisons, although the Table 5. Mate choice of the two male chubs O. evolans ♂Z. platypus ♂ Observed expected observed expected O. evolans ♀39 25.68 24 20.54 Z. platypus ♀1 14.32 8 11.46 p-value 1.12E-05**** 0.2025 ****p < 0.001. Table 6. Average numbers of satellites within a spawning event Type of mating pair Satellite number mean ± ste (min, max) O. evolans♂Z. platypus♂ O. evolans♂ × O. evolans♀0.95 ± 0.21 (0, 5) 0.23 ± 0.08 (0, 2) Z. platypus♂ × Z. platypus♀1.00 ± 0.33 (0, 2) 0.25 ± 0.25 (0, 2) Z. platypus♂ × O. evolans♀1.12 ± 0.21 (0, 4) 0.80 ± 0.18 (0, 3) Table 7. Satellite preference events Type of mating pair O. evolans satellite choice Z. platypus satellite choice Observed event Expected event Observed event Expected event O. evolans♂ × O. evolans♀18 (45%) 16.48 (41.2%) 8 (38%) 8.65 (41.2%) Z. platypus♂ × Z. platypus♀5 (12%) 5.12 (12.8%) 1 ( 5%) 2.69 (12.8%) Z. platypus♂ × O. evolans♀17 (43%) 9.20 (23%) 12 (57%) 4.83 (23%) O. evolans♂ × Z. platypus♀0 ( 0%) 9.20 (23%) 0 ( 0%) 4.83 (23%) Total number 40(100%) 40 (100%) 21(100%) 21 (100%) Table 8. Significance of O. evolans satellite choice among four kinds of mating pairs via Chi-square test p-value O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀ Z. platypus♂ × O. evolans♀ O. evolans♂ × Z. platypus♀ O. evolans♂ × O. evolans♀ Z. platypus♂× Z. platypus♀0.7053 Z. platypus♂ × O. evolans♀0.0094** 0.0101* O. evolans♂× Z. platypus♀0.0022*** 0.0024*** 0.0001*** *p < 0.05, **p < 0.01, ***p < 0.005. page 7 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan results were statistically significant (Tables 8 and 9). Thus, satellite males of both species might engage with their own species if they recognize mating males. DISCUSSION This study is the first to confirm that interspecific mating behavior frequently happens between introduced Z. platypus and native O. evolans (35% in Table 2). Previous studies indicated mixed genetic diversity, suggesting that introduced populations of Z. platypus hybridize with native populations in Lake Biwa (Takamura and Nakahara 2015) and very possibly with Taiwanese species in the Tamsui River (Ma et al. 2006). More and more hybrids of Z. platypus and distinct Nipponocypris species have been reported in Japan over the years (Arao and Shimoyama 2006; Katano et al. 2014; Kato 2004; Sakai et al. 1992). Thus, introduced Z. platypus may also hybridize with closely-related O. evolans and therefore decrease the populations of native chubs in Taiwan. A previous study indicated that Z. platypus females choose males based on the degree to which they express red nuptial coloration, and do not look for traits related to the greenish-blue nuptial color of males. Such female preference may be useful for selecting high-quality males with good foraging abilities or good immune functions related to carotenoids and sexual hormones (Takahashi 2018). Thus, closely-related O. evolans might have a similar preference mating. Statistical analysis further revealed that native O. evolans prefer native individuals (Table 3). In contrast, females of Z. platypus significantly prefer males of Z. platypus, across different relative proportions (Table 4). However, males of Z. platypus showed no significant preference for females of either species. Therefore, this might lead to interspecific mating pairs, which in turn would lead to hybrids (Table 5). This study implies that Z. platypus males may be unable to recognize conspecific females. Thus, introduced Z. platypus males compete with native O. evolans males for native females and reduce the successful production rate of native O. evolans. Meanwhile, introduced Z. platypus females also prefer to mate with males of Z. platypus. Thus, both situations may significantly decrease the populations of native O. evolans over time. Furthermore, satellite preference between the two species revealed that the satellite males of both species might engage with their own species because they recognize conspecific mating males. As a result, Z. platypus satellites mainly engage with Z. platypus♂ × O. evolans♀ mating pairs (Tables 6 and 9). Such satellite choice between the two species implies that Z. platypus males might be able to distinguish between males of two species, while O. evolans female cannot. In other words, Z. platypus male satellites might prefer to engage with Z. platypus while O. evolans might engage by chance. This bias may reduce the successful rate of O. evolans intraspecific mating, and this is supported by the observation that 35% of the spawning events were interspecific. Thus, if reproductive isolation remained in the two species and caused all hybrids to die, then the offspring number of O. evolans could decrease by 35% every generation. On the other hand, offspring of Z. platypus could have a higher chance of occupying O. evolans’ habitat niches. Therefore, one can expect a sequential decrease and serial increase in O. evolans and Z. platypus, respectively. CONCLUSIONS This study revealed that Z. platypus and putative hybrids might have colonized the Tamsui River as the result of previous fish fry releasing activities. Fortunately, records show that introduced Z. platypus only dispersed in the Tamsui River (Ma et al. 2006). To avoid genetic pollution from reoccurring introgression events and to maintain the genetic diversity of native Opsariichthys fishes, it is of the utmost importance that these Opsariichthys species are not artificially released into river systems. Further studies should focus on their population distribution, effective population Table 9. Significance of Z. platypus satellite choice among four kinds of mating pairs via Chi-square test p-value O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀ Z. platypus♂ × O. evolans♀ O. evolans♂ × Z. platypus♀ O. evolans♂ × O. evolans♀ Z. platypus♂ × Z. platypus♀0.2923 Z. platypus♂ × O. evolans♀0.0011*** 0.0006*** O. evolans♂ × Z. platypus♀0.0272* 0.0152* 0.0001*** *p < 0.05, ***p < 0.005. page 8 of 10Zoological Studies 59: 6 (2020) © 2020 Academia Sinica, Taiwan size, and threat to native species to understand future conservation biology strategies around speciation, hybridization, and genetic introgression between introduced Z. platypus and native Opsariichthys fishes. Acknowledgments: We thank Mr. Bo-Cyun Wang, Miss Chia-Yu Lin, and Miss Geng-Xin Kong for their field assistance, and Mr. Noah Last of Third Draft Editing for his English language editing. This study was funded by the Ministry of Science and Technology, Taiwan (MOST 105-2311-B-001-064, MOST 106-2311B-001-022, MOST 107-2311-B-001-007). Authors’ contributions: SPH and TYW designed the study and prepared the manuscript. NLL, SPH, and TYW performed the field work and video analysis. All authors participated in revising the manuscript and approved the final manuscript. Competing interests: NLL, SPH, and TYW declare that they have no conflict of interest. TYW has received research grants from the MOST, Taiwan. Availability of data and materials: There are two supplementary videos showing intraspecific and interspecific mating behaviors. supplement-vedio_cube. avi (video S1) was recorded on the underwater housing camera; supplement-vedio_handy-cam.avi (video S2) was recorded on the handy camera. Consent for publication: All authors have approved the manuscript and agree with its submission to Zoological Studies. Ethics approval consent to participate: Not applicable. 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